TWI894740B - Machine used for multi-surface biological sample imaging system, multi-surface biological sample imaging method, and non-transitory computer readable medium - Google Patents

Abstract

Biological samples on multiple surfaces of a support structure may be imaged using a machine comprising a lens, a flow cell and a controller. Such a machine may capture light emitted from nucleic acids disposed on first and second surfaces of the flow cell when the lens is, respectively, at first and second distances from the flow cell. In such a machine, the lens may be immersed in a first fluid, and the first and second surfaces of the flow cell may be separated by a second fluid. Additionally, in such a machine, differences between marginal and axial light rays in the field of view of the lens may be substantially equal when the lens is at the first and second distances from the flow cell.

Description

Apparatus for multi-surface biological sample imaging system, multi-surface biological sample imaging method, and non-transitory computer-readable medium

The disclosed technology relates to the field of imaging and evaluating analytical samples. More particularly, the disclosed technology relates to techniques for matching aberrations associated with imaging different surfaces of a multi-surface support structure.

There are an increasing number of applications for imaging analytical samples on support structures. Such support structures may include plates on which biological samples are present. For example, such plates may include deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) probes that are specific for nucleotide sequences present in the genes of humans and other organisms. Individual DNA or RNA probes can be attached to the support structure at specific locations in a small geometric grid or array. Depending on the technology used, samples can be attached to the support structure at random, semi-random, or predetermined locations. Test samples (e.g., from a known individual or organism) can be exposed to the array or grid, allowing complementary genes or fragments to hybridize to the probes at individual sites on the surface of the plate. In certain applications, such as sequencing, a template or fragment of genetic material can be located at a site, and nucleotides or other molecules can be hybridized with the template to determine the nature or sequence of the template. The site can then be examined by scanning light of a specific frequency over the site. Fluorescence at the site of hybridization allows identification of which genes or fragments are present in the sample.

Such plates are sometimes referred to as microarrays, gene or genomic chips, DNA chips, gene arrays, etc., and can be used for expression profiling, monitoring expression levels, genotyping, sequencing, etc. For example, diagnostic uses may include evaluating the genetic makeup of a particular patient to determine whether a disease state is present, or whether there is a predisposition to a particular condition. Reading and evaluating such plates is an important aspect of their utility. Although microarrays allow for the presence of independent biological components for bulk processing and individual detection, the number of components that can be detected in a single experiment is limited by the resolution of the system. In addition, the large amounts of reagents used in some methods can be expensive, making reduced volume desirable. While these issues can be addressed by increasing efficiency by imaging multiple surfaces of a single flow cell, multi-surface imaging can introduce further complications due to optical aberrations associated with each of the imaged surfaces. The disclosed technology provides methods and systems that enable highly efficient multi-surface array-based detection with lower cost and complexity than currently used methods and systems. Other advantages are also provided and will be apparent from the following description.

The disclosed technology provides a novel approach to imaging and evaluating analytical samples, reducing the complexity of imaging and evaluation subsystems with multiple surfaces supporting the samples. For example, the support structure can be a flow channel, which allows a reagent fluid to flow and interact with the biological sample. Excitation radiation from at least one radiation source can be used to excite the biological sample on multiple surfaces. In this manner, fluorescent radiation can be emitted from the biological sample and subsequently captured and detected by detection optics and at least one detector. The returned radiation can then be used to generate image data. This imaging of multiple surfaces can be performed sequentially or simultaneously. Furthermore, the technology described herein can be used with any of a variety of imaging systems. For example, epifluorescence and total internal reflection (TIR) methods can benefit from the disclosed technology. Furthermore, the biological samples being imaged can be present on the surface of the support structure in random locations or in a pattern.

One embodiment relates to a machine comprising a lens, a flow channel, and a controller. In this embodiment, the lens can have a field of view and be immersed in a first fluid having a first refractive index. Similarly, in this embodiment, the flow channel can include a first surface and a second surface separated by a second fluid having a second refractive index. In addition, the controller can move the lens from a first position having a first distance from the flow channel to a second position having a second distance from the flow channel. When the lens is separated from the flow channel by the first distance, the controller can also use the lens to capture light emitted by nucleic acids disposed on the first surface of the flow channel. When the lens is separated from the flow channel by the second distance, the controller may also use the lens to capture light emitted by nucleic acids disposed on the second surface of the flow channel. The controller may also determine the nucleic acid sequence of a biological sample based on the emitted light. In this embodiment, when the lens is separated from the flow channel by the first distance, the optical path difference between marginal light and axial light in the field of view of the lens may be substantially equal to the optical path difference between marginal light and axial light in the field of view of the lens when the lens is separated from the flow channel by the second distance.

In some embodiments of a machine, as described in the previous paragraph, the first refractive index and the second refractive index can be substantially equal.

In some embodiments of an apparatus, as described in the preceding paragraph, the lens may be comprised of an optical device adapted to capture light emitted by nucleic acid sequences disposed on the first surface of the flow cell and allow the biological sample to be imaged on a detector with diffraction-limited imaging quality. In some such embodiments, the first refractive index and the second refractive index are substantially equal, meaning that any spherical aberration caused by any difference between the first and second refractive indices is sufficiently low as not to prevent diffraction-limited imaging of the biological sample based on: light emitted by nucleic acid sequences disposed on the first surface of the flow cell; and light emitted by nucleic acid sequences disposed on the second surface of the flow cell.

In some embodiments of a machine, as described in the third paragraph of the present invention, the first fluid and the second fluid may be the same fluid.

In some embodiments of a machine, as described in the third paragraph of the present invention, the first fluid and the second fluid may be different fluids.

In some embodiments of a machine, as described in the second paragraph of the present disclosure, the first surface of the flow channel is a top surface of the flow channel, and the second surface of the flow channel is a bottom surface of the flow channel.

In some embodiments of an apparatus, as described in the second paragraph of the present invention, the lens may be comprised of an optical device adapted to capture light emitted by nucleic acid sequences disposed on the first surface of the flow cell and allow the biological sample to be imaged on a detector with diffraction-limited imaging quality. In some such embodiments, the optical path difference between marginal and axial rays in the field of view of the lens when the lens is separated from the flow cell by the first distance is substantially equal to the difference between marginal and axial rays in the field of view of the lens when the lens is separated from the flow cell by the second distance, meaning that spherical aberration is sufficiently low so as not to prevent diffraction-limited imaging of the biological sample based on: light emitted by nucleic acid sequences disposed on the first surface of the flow cell; and light emitted by nucleic acid sequences disposed on the second surface of the flow cell.

Another embodiment relates to a method comprising: capturing light emitted by nucleic acid disposed on a first surface of a flow channel using a lens, the lens being located a first distance from the flow channel and immersed in a first fluid having a first refractive index. The method may also include moving the lens to a position a second distance from the flow channel. The method may also include capturing light emitted by nucleic acid disposed on a second surface of the flow channel using the lens, the lens being immersed in the first fluid having the first refractive index, while the lens is located the second distance from the flow channel, wherein the first surface of the flow channel and the second surface of the flow channel are separated by a second fluid having a second refractive index. The method may also include determining a nucleic acid sequence in a biological sample based on light emitted by nucleic acids disposed on the first surface of the flow channel and light emitted by nucleic acids disposed on the second surface of the flow channel. In this method, when the lens is at the first distance from the flow channel, the optical path difference between marginal light and axial light in the field of view of the lens is substantially equal to the optical path difference between marginal light and axial light in the field of view of the lens when the lens is at the second distance from the flow channel.

In some embodiments of a method, as described in the previous paragraph, the first refractive index and the second refractive index can be substantially equal.

In some embodiments of a method, as described in the previous paragraph, the lens can be comprised of an optical device adapted to capture light emitted by nucleic acid sequences disposed on the first surface of the flow cell and allow the biological sample to be imaged on a detector with diffraction-limited imaging quality. In some such embodiments, the first refractive index and the second refractive index are substantially equal, meaning that any spherical aberration caused by any difference between the first and second refractive indices is sufficiently low so as not to prevent diffraction-limited imaging of the biological sample based on: light emitted by nucleic acid sequences disposed on the first surface of the flow cell; and light emitted by nucleic acid sequences disposed on the second surface of the flow cell.

In some embodiments of a method, as described in paragraph 10 of the present invention, the first fluid and the second fluid may be the same fluid.

In some embodiments of a method, as described in paragraph 10 of the present invention, the first fluid and the second fluid may be different fluids.

In some embodiments of a method, as described in paragraph 9 of the present disclosure, the first surface of the flow channel is a top surface of the flow channel, and the second surface of the flow channel is a bottom surface of the flow channel.

In some embodiments of a method, as described in paragraph 9 of the present invention, the lens is comprised of an optical device adapted to capture light emitted by nucleic acid sequences disposed on the first surface of the flow cell and allow the biological sample to be imaged on a detector with diffraction-limited imaging quality. In some such embodiments, the optical path difference between marginal and axial rays in the field of view of the lens when the lens is at the first distance from the flow cell is substantially equal to the optical path difference between marginal and axial rays in the field of view of the lens when the lens is at the second distance from the flow cell, meaning that spherical aberration is sufficiently low so as not to prevent diffraction-limited imaging of the biological sample based on: light emitted by nucleic acid sequences disposed on the first surface of the flow cell; and light emitted by nucleic acid sequences disposed on the second surface of the flow cell.

Another embodiment relates to a non-transitory computer-readable medium storing instructions that, when executed by a processor, cause a biological sample imaging system to perform actions. In some such embodiments, the actions may include capturing light emitted by nucleic acids disposed on a first surface of a flow cell using a lens, the lens being at a first distance from the flow cell, the lens having a field of view, and immersed in a first fluid having a first refractive index. In some such embodiments, the actions may include moving the lens to a position at a second distance from the flow cell. In some such embodiments, the actions may include capturing light emitted by nucleic acids disposed on a second surface of the flow cell using the lens, the lens being immersed in the first fluid having the first refractive index, and the lens being located at the second distance from the flow cell, wherein the first surface of the flow cell and the second surface of the flow cell are separated by a second fluid having a second refractive index. In some such embodiments, the actions may include determining a nucleic acid sequence in a biological sample based on the light emitted by the nucleic acids disposed on the first surface of the flow cell and the light emitted by the nucleic acids disposed on the second surface of the flow cell. In some such embodiments, the optical path difference between marginal rays and axial rays in the field of view of the lens when the lens is at the first distance from the flow channel can be substantially equal to the optical path difference between marginal rays and axial rays in the field of view of the lens when the lens is at the second distance from the flow channel.

In some embodiments, as described in the previous paragraph, the first refractive index and the second refractive index can be substantially equal.

In some embodiments, as described in the previous paragraph, the lens can be comprised of an optical device adapted to capture light emitted by nucleic acid sequences disposed on the first surface of the flow cell and allow the biological sample to be imaged on a detector with diffraction-limited imaging quality. In some such embodiments, the first refractive index and the second refractive index are substantially equal, meaning that any spherical aberration caused by any difference between the first and second refractive indices is sufficiently low so as not to prevent diffraction-limited imaging of the biological sample based on: light emitted by nucleic acid sequences disposed on the first surface of the flow cell; and light emitted by nucleic acid sequences disposed on the second surface of the flow cell.

In some embodiments, as described in paragraph 16 of the present disclosure, the first surface of the flow cell is a top surface of the flow cell, and the second surface of the flow cell is a bottom surface of the flow cell. In some embodiments, as described in paragraph 16 of the present disclosure, the lens is comprised of an optical device adapted to capture light emitted by nucleic acid sequences disposed on the first surface of the flow cell and allow the biological sample to be imaged on a detector with diffraction-limited imaging quality. In some such embodiments, the optical path difference between marginal and axial rays in the field of view of the lens when the lens is at the first distance from the flow cell is substantially equal to the optical path difference between marginal and axial rays in the field of view of the lens when the lens is at the second distance from the flow cell, meaning that spherical aberration is sufficiently low so as not to prevent diffraction-limited imaging of the sample based on: light emitted by nucleic acid sequences disposed on the first surface of the flow cell; and light emitted by nucleic acid sequences disposed on the second surface of the flow cell.

In some embodiments, as described in paragraph 16 of the present invention, the first fluid and the second fluid may be the same fluid.

Other features and aspects of the disclosed technology will become apparent from the following embodiments in conjunction with the accompanying drawings. For example, the accompanying drawings illustrate features of embodiments according to the disclosed technology. The present disclosure is not intended to limit the scope of any protection provided by this document or any related documents, which is defined by the scope of the respective patent applications and equivalents.

It should be understood that all combinations of the aforementioned concepts (assuming such concepts are not mutually inconsistent) are contemplated as part of the inventive subject matter disclosed herein. Specifically, all combinations of claimed subject matter appearing at the end of this disclosure are contemplated as part of the inventive subject matter disclosed herein.

10: Biological Sample Imaging System

12: Biological components

14: Biological components

16: Support structure

18: Surface

20: Surface

22,24: Radiation Source

26,28: Adjusting optical devices

30: Guiding Optical Devices

32: Focusing Optics

34: Detection Optical Devices

36: Detector

38: Control/Processing System

40: Translation system

42: Upper board; lower board; first board

44: Lower board; second board

46: Internal volume; support structure

48: Stimulate Radiation

50: Radiation line

52: Arrow

54: Area

56: Attachment layer

58: First stimulus radiation source; source; stimulus source

60: First fluorescent emission

62: Second source of stimulated radiation; source

64: Second fluorescent emission

66: Spatially ordered pattern; fixed array

68: Biological component location

70: Random spatial distribution; random distribution

72: Biological component location

74: Plate N-2

76: Plate N-1

78: Plate N

80: M-3 Surface

82: Surface M-2

84: Surface M-1

86: Mth Surface

88: Radiation Source

90: Fluorescent emission

92:Objective lens

801: Top surface

802: Bottom surface

Figure 1 is a schematic overview of a biological specimen imaging system. Figure 2 is a schematic perspective view of exemplary radiation directed toward the surface of a support structure to semi-confocally illuminate a biological site and return the radiation semi-confocally to a detector. Figure 3 is a cross-sectional view of an exemplary support structure in which excitation radiation is directed to two surfaces of the support structure. Figure 4 is a schematic perspective view of an exemplary support structure having an array of biological component sites in a spatially ordered pattern. Figure 5 is a schematic perspective view of an exemplary support structure having an array of biological component sites in a spatially ordered pattern. A schematic perspective view of an exemplary support structure with randomly spatially distributed biological component locations; [Figure 6] is a cross-sectional view of an exemplary support structure in which excitation radiation is directed to multiple surfaces of the support structure; [Figure 7] illustrates exemplary dimensions between the objective lens and the support structure that may exist in some embodiments; [Figure 8] illustrates exemplary ray tracing from a light source to the first lens of an objective lens for imaging multiple surfaces; and [Figure 9] illustrates exemplary images expected from imaging the top and bottom surfaces when the imaging system is optimized for imaging the top surface.

Turning now to the drawings, and referring initially to FIG. 1 , a biological sample imaging system 10 is schematically illustrated. Biological sample imaging system 10 is capable of imaging multiple biological components 12, 14 within a support structure 16. For example, in the illustrated embodiment, a first biological component 12 may be present on a first surface 18 of support structure 16, while a second biological component 14 may be present on a second surface 20 of support structure 16. For example, support structure 16 may be a flow channel having an array of biological components 12, 14 on interior surfaces 18, 20. These surfaces are generally facing each other, and reagents, rinse solutions, and other fluids may be introduced through these surfaces, e.g., to bind nucleotides or other molecules to sites on biological components 12, 14. Fluorescent labels attached to molecules of the components can include, for example, dyes that fluoresce when excited by appropriate excitation radiation. Analytical methods that include the use of fluorescent labels and can be used in the apparatus or methods described herein include those described elsewhere herein, such as genotyping analysis, gene expression analysis, methylation analysis, or nucleic acid sequencing analysis.

Those skilled in the art will appreciate that the flow cell can be used with any of a variety of arrays known in the art to achieve similar results. Such arrays can be formed by placing biological components of a sample on a support surface, either randomly or in a predetermined pattern, using a variety of techniques. In a specific embodiment, a clustered array of nucleic acid clones can be prepared as described in U.S. Patent No. 7,115,400; U.S. Patent Application Publication No. 2005/0100900; PCT Publication No. WO 00/18957; or PCT Publication No. WO 98/44151, each of which is incorporated herein by reference. This type of approach is called bridge amplification or solid-phase amplification and is particularly well-suited for sequencing applications.

Other exemplary random arrays that can be used include, but are not limited to, random arrays of beads associated with a solid support, examples of which are described in U.S. Patent Nos. 6,355,431; 6,327,410; and 6,770,441; U.S. Patent Application Publication Nos. 2004/0185483 and 2002/0102578; and PCT Publication No. WO 00/63437, each of which is incorporated herein by reference. The beads can be located at discrete locations on the solid support, such as wells, with each location housing a single bead.

Any of a variety of other arrays known in the art, or methods for making such arrays, may be used. Commercially available microarrays that may be used include, for example, Affymetrix® GeneChip® microarrays or microarrays based on what is sometimes referred to as VLSIPS ^™ (Very Large Scale Immobilized Polymer Synthesis). Other microarrays synthesized using the technology of Synthesis) are described in the following references: U.S. Patent Nos. 5,324,633; 5,744,305; 5,451,683; 5,482,867; 5,491,074; 5,624,711; 5,795,716; 5,831,070; 5,856,101; 5,858,659; 5,874,219; 5,968,740; Nos. 5,974,164; 5,981,185; 5,981,956; 6,025,601; 6,033,860; 6,090,555; 6,136,269; 6,022,963; 6,083,697; 6,291,183; 6,309,831; 6,416,949; 6,428,752; and 6,482,591, each of which is incorporated herein by reference. Spotted microarrays may also be used. An exemplary spotted microarray is the CodeLink ^™ Array available from Amersham Biosciences. Another suitable microarray is one manufactured using inkjet printing methods, such as SurePrint ^™ Technology available from Agilent Technologies.

The sites or features of an array are typically discrete, separated by space. The size of the sites and/or the spacing between sites can vary. For example, arrays useful in conjunction with the disclosed techniques may have sites separated by less than 100 μm, 50 μm, 10 μm, 5 μm, 1 μm, or 0.5 μm (e.g., 350 nm or 10 nm). Apparatus or methods based on embodiments of the present disclosure can be used to image the array with a resolution sufficient to distinguish sites within such densities or density ranges.

As exemplified herein, the surfaces used in the apparatus or methods according to the present disclosure are generally artificial surfaces. Natural surfaces or surfaces of natural supporting structures may also be used; however, the disclosed techniques can be applied to embodiments involving surfaces of non-natural materials or surfaces of non-natural supporting structures. Thus, components of a biological sample can be removed from their natural environment and attached to an artificial surface.

Any of a variety of biological components may be present on a surface. Exemplary components include, but are not limited to, nucleic acids (such as DNA or RNA), proteins (such as enzymes or receptors), polypeptides, nucleotides, amino acids, carbohydrates, cofactors, or metabolites or derivatives of these naturally occurring components. Although the apparatus and methods described herein are illustrated using components of biological samples, it should be understood that other samples or components may also be used. For example, synthetic samples such as combinatorial libraries or compound libraries containing species known or suspected to have a desired structure or function may be used. Thus, the apparatus or methods can be used to synthesize a collection of compounds and/or screen a collection of compounds for a desired structure or function.

Returning to the exemplary system of FIG1 , the biological sample imaging system 10 may include at least a first radiation source 22 and may also include a second radiation source 24 (or additional radiation sources). The radiation sources 22, 24 may be lasers operating at different wavelengths. The choice of wavelength for the laser is generally determined by the fluorescent properties of the dye used to image the component site. Using multiple lasers of different wavelengths allows the dye at the same site within the branching structure 16 to be distinguished, and imaging can be performed by continuously acquiring a series of images to achieve identification of the molecule at the component site based on image processing and readout logic generally known in the art. In other embodiments, light emitting diodes (LEDs) may be used as the radiation sources 22, 24. Other radiation sources known in the art may be used, including, for example, arc lamps or quartz halogen lamps. Particularly suitable radiation sources are those that generate electromagnetic radiation in the ultraviolet (UV) range (approximately 200 to 390 nm), the visible (VIS) range (approximately 390 to 770 nm), the infrared (IR) range (approximately 0.77 to 25 microns), or other regions of the electromagnetic spectrum.

For ease of description, embodiments based on fluorescence detection are used as examples. However, it should be understood that other detection methods can be used in conjunction with the apparatus and methods described herein. For example, a variety of emission types can be detected, such as fluorescence, luminescence, or chemiluminescence. Thus, the component to be detected can be labeled with a fluorescent, luminescent, or chemiluminescent compound or moiety. Signals other than optical signals can also be detected from various surfaces using apparatus and methods similar to those exemplified herein, but modified to accommodate the specific physical properties of the signal to be detected.

The output from radiation sources 22 and 24 can be directed through conditioning optics 26 and 28 to filter and shape the beam. For example, in a presently contemplated embodiment, conditioning optics 26 and 28 can produce a substantially linear radiation beam and combine beams from multiple lasers, as described, for example, in U.S. Patent No. 7,329,860. The laser module can additionally include a measurement component that records the power of each laser. The power measurement can be used as a feedback mechanism to control the duration of image recording to obtain a uniform exposure and, therefore, a more easily comparable signal. In other embodiments, conditioning optics 26 and 28 can form a rectilinear radiation beam, such as a square or rectangular shape.

After passing through the conditioning optics 26, 28, the beam can be directed toward the steering optics 30, which redirects the beam from the radiation sources 22, 24 toward the focusing optics 32. The steering optics 30 can include a dichroic mirror configured to redirect the beam toward the focusing optics 32 while also allowing certain wavelengths of a retrobeam to pass through. The focusing optics 32 can confocally direct the radiation toward one or more surfaces 18, 20 of the support structure 16, where the individual biological components 12, 14 are located. For example, the focusing optics 32 can include a microscope objective that confocally directs the radiation sources 22, 24 along a line and focuses them onto the surfaces 18, 20 of the support structure 16. In some embodiments, focusing optics 32 can focus radiation sources 22, 24 above or below surfaces 18, 20 to a concentration below a saturation threshold for the species to be excited and/or below a threshold for minimizing damage to the sample. In such an embodiment, focusing optics 32 can also maintain the focal plane of detector 36 in the plane of surfaces 18, 20 when the excitation radiation is out of focus relative to the plane of surfaces 18, 20. In some embodiments, focusing optics 32 can be moved relative to surfaces 18, 20. For example, focusing optics 32 can be mounted to a z-stage, such as a voice coil or piezoelectric assembly, to move focusing optics 32 along an axis perpendicular to the plane of surfaces 18, 20. In some embodiments, focusing optics 32 can be moved relative to surfaces 18, 20 along the x-axis and/or the y-axis.

The biological component sites on the support structure 16 can utilize one or more reagents that fluoresce at specific wavelengths in response to the excitation beam and thus return radiation for imaging. For example, fluorescent components can be generated by hybridizing fluorescently labeled nucleotides with complementary nucleotides in the sample using a polymerase. In other embodiments, fluorescently labeled nucleotides can be temporarily associated with complementary nucleotides in the sample and subsequently removed. In yet further embodiments, fluorescently labeled oligonucleotides can be used. As mentioned above, the fluorescent properties of these components can be altered by introducing reagents into the support structure 16 (e.g., by cleaving the dye from the molecule, preventing the attachment of additional molecules, adding quenching reagents, adding energy transfer acceptors, etc.). As one skilled in the art will appreciate, the wavelength at which the sample's dye is excited, and therefore the wavelength at which it fluoresces, will depend on the absorption and emission spectra of the particular dye. This return radiation can propagate back through the guidance optics 30. This reflected beam can typically be directed toward detection optics 34, which can filter the beam, for example, to separate different wavelengths within the reflected beam, and direct the reflected beam toward at least one detector 36.

The detector 36 can be based on any suitable technology and can be, for example, a charge coupled device (CCD) sensor that generates pixelated image data based on the position of a photon impacting the device. However, it should be understood that any of a variety of other detectors may be used, including but not limited to a detector array configured for time delay integration (TDI) operation, a complementary metal oxide semiconductor (CMOS) detector, an avalanche photodiode (APD) detector, a Geiger-mode photon counter, or any other suitable detector. TDI mode detection can be coupled with line scanning, as described in U.S. Patent No. 7,329,860.

The detector 36 can generate image data, for example, at a resolution between 0.1 and 50 microns, which is then transferred to a control/processing system 38. Generally, the control/processing system 38 can perform various operations, such as analog-to-digital conversion, scaling, filtering, and correlating the data in multiple frames, to appropriately and accurately image multiple sites at specific locations on the sample. The control/processing system 38 can store the image data and can ultimately transfer the image data to a post-processing system (not shown) that analyzes the data. Depending on the type of sample, the reagents used, and the processing performed, the image data can be used in a variety of different ways. For example, nucleotide sequence data can be derived from the image data, or the data can be used to determine the presence of a specific gene, characterize one or more molecules at a component site, etc. The operation of the various components depicted in FIG1 may also be coordinated with a control/processing system 38. In practical applications, the control/processing system 38 may include hardware, firmware, and software designed to control the operation of the radiation sources 22 and 24, the movement and focusing of the focusing optics 32, the translation system 40, and the detection optics 34, as well as the acquisition and processing of signals from the detector 36. The control/processing system 38 may thus store processed data and further process the data to generate a reconstructed image of the irradiated locations within the support structure 16 that emitted fluorescence. The image data may be analyzed by the system itself or may be stored for analysis by other systems and at different times after imaging.

Support structure 16 can be supported on a translation system 40 that allows for focusing and movement of support structure 16 before and during imaging. The stage can be configured to move support structure 16, thereby changing the relative positions of radiation sources 22, 24 and detectors 36 relative to the surface to which the biological component is bound, for progressive scanning. Translation system 40 can move in one or more dimensions, including, for example, one or both dimensions orthogonal to the direction of propagation of the excitation radiation, commonly designated as the X and Y dimensions. In certain embodiments, translation system 40 can be configured to move in a direction perpendicular to the scanning axis of a detector array. The translation system 40 can be further configured to move in a dimension along which the excitation radiation propagates, typically denoted as the Z dimension. Movement in the Z dimension can also be used for focusing.

FIG2 is a diagrammatic representation of an exemplary semi-confocal line scanning method for imaging the support structure 16. In the depicted embodiment, the support structure 16 includes an upper plate 42 and a lower plate 44 with an internal volume 46 therebetween. The upper plate 42 and the lower plate 44 can be made of any of a variety of materials, but are preferably made of a substrate material that is substantially transparent at the wavelengths of the excitation radiation and the fluorescent return beam, allowing the excitation radiation and the returned fluorescent emission to pass through without significant signal quality loss. Furthermore, when used in an epifluorescent imaging configuration as shown, one of the surfaces penetrated by the radiation can be substantially transparent at the relevant wavelength, while the other surface (not penetrated by the radiation) can be relatively opaque, semi-transparent, or even opaque or reflective. The upper and lower plates 42, 44 can comprise a polymeric material or other intermediate component, and both can further contain biological components 12, 14 on their respective inwardly facing surfaces 18, 20. As discussed above, the internal volume 46 can, for example, include one or more internal passageways of a flow channel through which a reagent fluid can flow.

Reactive elements (e.g., fluorinated nucleotides) associated with support structure 16 can be irradiated by excitation radiation 48 along radiation line 50. Radiation line 50 can be formed by excitation radiation 48 from radiation sources 22, 24, directed by guiding optics 30 through focusing optics 32. Radiation sources 22, 24 can generate a beam that is processed and shaped to provide a linear cross section or line of radiation comprising a plurality of wavelengths of radiation to cause fluorescence from biological components 12, 14 at corresponding wavelengths (depending on the particular dye used). Next, focusing optics 32 can semi-confocally direct excitation radiation 48 toward first surface 18 of support structure 16 to irradiate fluorinated nucleotide sites associated with biological component 12 along radiation ray 50. Furthermore, support structure 16, guiding optics 30, focusing optics 32, or some combination thereof can be slowly translated so that the generated radiation ray 50 progressively irradiates the components as indicated by arrow 52. This translation results in a continuous scan of region 54, which allows fluorinated nucleotides associated with biological component 12 to be progressively irradiated across first surface 18 of support structure 16. As discussed in more detail below, the same procedure can also be used to progressively irradiate second surface 20 of support structure 16. In practice, this procedure can be used for multiple surfaces within the support structure 16.

Exemplary methods and apparatus for line scanning are described in U.S. Patent No. 7,329,860, which is incorporated herein by reference, and which describes a line scanning apparatus having a detector array configured to achieve confocality along the scan axis by limiting the scan axis dimension of the detector array. More specifically, the scanning apparatus can be configured such that the detector array has rectangular dimensions, such that the shorter dimension of the detectors is within the scan axis dimension, and the imaging optics are positioned to direct a rectangular image of a sample area onto the detector array such that the shorter dimension of the image is also within the scan axis dimension. Because confocality occurs along a single axis (i.e., the scanning axis), semi-confocality is achieved in this manner. Consequently, detection is limited to features on the substrate surface, excluding signals that may arise from the solution surrounding the features. The apparatus and method described in U.S. Patent No. 7,329,860 can be modified to scan two or more supported surfaces as described herein.

Detection devices and methods other than line scanning may also be used. For example, point scanning may be used as described below or in U.S. Patent No. 5,646,411, which is incorporated herein by reference. Wide-angle area detection may be used with or without scanning motion. As described in more detail elsewhere herein, TIR methods may also be used.

As generally illustrated in FIG2 , the radiation line 50 used to image the sites of the biological components 12 and 14 can be a continuous line or a discontinuous line. Thus, some embodiments may include a discontinuous line composed of a plurality of confocally or semi-confocally directed radiation beams that still irradiate a plurality of sites along the radiation line 50. These discontinuous beams can be generated by one or more sources that are positioned or scanned to provide the excitation radiation 48. As previously described, these beams can be directed confocally or semi-confocally toward the first surface 18 or the second surface 20 of the support structure 16 to irradiate the sites of the biological components 12 and 14. As described above for continuous semi-confocal line scanning, the support structure 16, guiding optics 30, focusing optics 32, or some combination thereof can be slowly advanced as indicated by arrow 52 to irradiate a continuously scanned area 54 along the first surface 18 or the second surface 20 of the support structure 16, thereby irradiating a continuous area of the biological components 12, 14.

It should be noted that the system typically forms and directs both the excitation radiation and the return radiation simultaneously for imaging. In some embodiments, confocal point scanning can be used so that the optical system directs the excitation point or spot across the biological component by scanning the excitation beam through the objective lens. The detection system can image the emission from the excitation point on the detector without having to "descan" the return beam. This is because the return beam is collected by the objective lens and separated from the excitation beam optical path before returning through the scanning component. Therefore, depending on the field angle of the original excitation spot in the objective lens, the return beam will appear on the detector 36 at different points. As the excitation spot is scanned across the sample, its image on detector 36 appears as a straight line. This configuration is useful, for example, if the scanning module cannot, for some reason, receive a reflected beam from the sample. Examples include holographic and acousto-optical scanning modules, which can scan beams at very high speeds but use diffraction to produce the scan. Therefore, the scanning properties vary with wavelength. The reflected beam of emitted radiation has a different wavelength than the excitation beam. Alternatively or additionally, the emission signal can be collected sequentially after consecutive excitations at different wavelengths.

In certain embodiments, an apparatus or method may detect features on a surface at a rate of at least about 0.01 ^mm² /sec. Depending on the particular application, faster rates may also be used, including, for example, at least about 0.02 ^mm² /sec, 0.05 ^mm² /sec, 0.1 ^mm² /sec, 1 ^mm² /sec, 1.5 ^mm² /sec, 5 ^mm² /sec, 10 mm²/sec, 50 ^mm² /sec, ^{100 mm² /} sec, or faster rates, depending on the area scanned or otherwise detected. If necessary (eg, to reduce noise), the detection rate may have an upper limit of approximately 0.05 mm ² /sec, 0.1 ^{mm 2} /sec, 1 mm ² /sec, 1.5 mm ² / ^sec , 5 mm 2 /sec, 10 mm ² /sec, 50 mm ² /sec, or 100 mm ² /sec.

In some cases, the support structure 16 can be used in a manner such that the biological component is intended to be present on only one surface. However, in some cases, the biological material is present on multiple surfaces within the support structure 16. For example, FIG3 illustrates a typical support structure 16 in which the biological material has been attached to both the first surface 18 and the second surface 20. In the illustrated embodiment, an attachment layer 56 has been formed on both the first surface 18 and the second surface 20 of the support structure 16. A first excitation radiation source 58 can be used to irradiate one of the plurality of sites of the biological component 12 on the first surface 18 of the support structure 16 and return a first fluorescent emission 60 from the fluorinated nucleotide associated with the irradiated biological component 12. Simultaneously or sequentially, a second excitation radiation source 62 can be used to irradiate one of a plurality of sites on the biological component 14 on the second surface 20 of the support structure 16 and return a second fluorescent emission 64 from the irradiated biological component 14.

While the embodiment illustrated in FIG3 shows excitation from sources 58 and 62 from the same side of support structure 16, it should be understood that the optical system can be configured to illuminate surfaces on opposite sides of support structure 16. Taking FIG3 as an example, as shown, upper surface 18 can be irradiated from first excitation source 58, and lower surface 20 can be irradiated from below. Similarly, emissions can be detected from one or more sides of the support structure. In certain embodiments, different sides of support structure 16 can be excited from the same radiation source by first irradiating one side and then flipping the support structure so that the other side is positioned for excitation by the radiation source.

The distribution of biological components 12, 14 can follow many different patterns. For example, FIG4 illustrates a support structure 16 in which biological components 12, 14 are uniformly distributed at sites or features on first and second surfaces 18, 20 in a spatially ordered pattern 66 of biological component sites 68. For example, certain types of microarrays can be used in which the positions of individual biological component sites 68 can follow a regular spatial pattern, such as a hexagonal pattern. The pattern can include sites at predefined locations. In contrast, in other types of bioimaging arrays, biological components are attached to a surface at randomly occurring sites or statistically varying locations, allowing imaging of the microarray to determine the location of each of the individual biological component features. Thus, the pattern of features need not be predetermined, despite being a product of a synthetic or fabrication process.

For example, FIG5 illustrates a support structure 16 in which sites on the first surface 18 and the second surface 20 are located within a random spatial distribution 70 of biological component sites 72. However, it is possible to image multiple surfaces 18, 20 of a support structure 16 having both fixed arrays 66 and random distributions 70 of biological sample sites. Furthermore, it should be noted that in both cases, the biological component at each site can consist of either a population of identical molecules or a random mixture of different molecules. Furthermore, the density of the biological sample can vary and can be at least 1,000 sites per square millimeter.

The present technology can accommodate such different physical configurations of multiple surfaces within the support structure 16, as well as different arrangements of sites within the components on the surfaces. As described above with reference to Figures 2 and 3 , in an embodiment of a support structure 16 having a first surface 18 and a second surface 20, a first excitation radiation source 58 can illuminate sites of biological components 12 on the first surface 18 and return a first fluorescent emission 60 ; simultaneously, a second excitation radiation source 62 can illuminate sites of biological components 14 on the second surface 20 and return a second fluorescent emission 64 , as illustrated in Figure 3 . Therefore, detecting the components of the volume of the sample between the two surfaces is unnecessary and can be eliminated. Selective detection of a surface of a supporting structure provides for preferential detection of the surface relative to the volume of the supporting structure adjacent to the surface, and relative to one or more other surfaces of the supporting structure.

In more complex configurations, irradiating more than two surfaces is suitable. For example, Figure 6 shows a support structure 16 having N plates, including a first plate 42, a second plate 44, ..., an (N-2)th plate 74, an (N-1)th plate 76, and an (N)th plate 78. These plates define M surfaces, including a first surface 18, a second surface 20, ..., an (M-3)th surface 80, an (M-2)th surface 82, an (M-1)th surface 84, and an (M)th surface 86. In the illustrated embodiment, not only the first surface 18 and the second surface 20 of the support structure 16 can be irradiated, but all M surfaces can be irradiated. For example, an excitation radiation source 88 can be used to irradiate biological component sites on the Mth surface 86 of the support structure 16 and return a fluorescent emission 90 from the irradiated biological component. For support structures with multiple surfaces, it is preferred to excite the upper layer from the top and the lower layer from the bottom to reduce photobleaching. Thus, components on a layer closer to a first outer side of the support structure can be irradiated from the first side, while irradiation from the opposite outer side can be used to excite components present on layers closer to the opposite outer side.

FIG7 illustrates an objective lens 92 through which radiation from emissive biological components 12, 14 located on the first surface 18 and the second surface 20, respectively, of the support structure 16 can be detected. The objective lens 92 can be one of the components of the focusing optical device 32 described above. Although not drawn to scale, FIG7 illustrates exemplary dimensions between the objective lens 92 and the support structure 16. For example, the objective lens 92 can be spaced approximately 500 microns from the upper plate 42 of the support structure 16. The biological specimen imaging system 10 can be configured to detect emitted radiation from the biological component 12 on the first surface 18 through the upper plate 42, which can be made of, for example, glass having a thickness of 300 μm to 700 μm. Furthermore, the biological sample imaging system 10 can also be configured to detect radiation emitted from the biological components 14 on the second surface 20 through a fluid (e.g., a reagent solution, such as a buffer solution, imaging solution, or other reagent) within the interior volume 46 of the upper plate 42 (which can be, for example, 300 to 700 μm) plus the support structure 46. The thickness of the interior volume can vary from 75 to 100 μm, as shown.

In some embodiments, objective lens 92 may be a plano-convex (PCX) lens, with its flat surface facing the sample to avoid turbulence in the liquid flow when the objective is used for liquid immersion imaging. In such embodiments, objective lens 92 can be immersed in the fluid, which may allow it to have a significantly higher numerical aperture compared to dry objectives, as the numerical aperture is linearly proportional to the refractive index of the medium surrounding the objective. This, in turn, can increase the throughput of systems based on embodiments of the present disclosure, as the throughput is proportional to the square of the numerical aperture.

While liquid immersion allows for an increase in numerical aperture, it also increases aberrations such as wavefront errors (e.g., spherical aberration) when performing multi-surface imaging. This can be seen in Figure 8, which shows ray tracing from the light source to the first lens of the objective for imaging the top surface 801 and the bottom surface 802 in a dual-surface imaging configuration. As shown, the length of the segment _{Mc Mi} on the marginal rays increases with angle θ _i in both top and bottom surface imaging. Therefore, since the maximum wavefront error can be evaluated as the optical path difference between the axial and marginal rays, the greater the refractive index of the medium separating the objective and the top surface ( _ni ), the greater the angle θ _i , and therefore the greater the error in the ordering using the depicted optical layout.

Although liquid immersion increases wavefront error, this error is predictable and can be compensated for through the design of objective lens 92. For example, by designing the objective lens to compensate for the expected wavefront error in imaging the top surface, a diffraction-limited quality image of the top surface can be obtained. However, as shown in FIG8 , since the optical paths traveled by light differ when imaging different surfaces of support structure 16, optimizing the image of first surface 18 results in a distortion of the image from second surface 20. This is illustrated graphically in FIG9 , which depicts exemplary images of the first surface 18 and second surface 20 of the support structure 16 corresponding to the thickness of the upper plate 42 (e.g., 300 microns) in the configuration shown in FIG7 , where the imaging system is optimized for first surface 18. As shown, the imaging system is capable of providing high image quality on the first surface 18 because, in the example of FIG9 , it is designed to do so. However, in a system optimized for imaging from the first surface, images captured from the second surface 20 may contain aberrations. Similarly, if the system is optimized for imaging from the second surface 20, images captured from the first surface 18 may contain aberrations.

The errors that may be encountered when imaging from one surface by a system optimized for imaging from different surfaces are expressed mathematically in Equations 1 to 3 below. In those equations, W _top is the wavefront error at the top surface, W _bottom is the wavefront error at the bottom surface, n _c is the refractive index of the cover glass, n _i is the refractive index of the immersion fluid, n _g is the refractive index of the first lens of the objective lens, n _b is the refractive index of the reagent solution, θ _c is the angle between the marginal ray and the axial ray passing through the cover glass, θ _i is the angle between the marginal ray and the axial ray passing through the immersion fluid, θ _g is the angle between the marginal ray and the axial ray passing through the first lens of the objective lens, θ _b is the angle between the marginal ray and the axial ray passing through the reagent solution, t _c is the thickness of the cover glass, _ti is the thickness of the immersion fluid used to image the top surface, and _ti is the thickness of the immersion fluid used for bottom surface imaging, _tg is the thickness of the first lens of the objective, _tb is the thickness of the reagent solution, and Δ is the difference in wavefront error between the top and bottom surfaces (e.g., the aberration associated with bottom surface imaging in a system optimized for top surface imaging).

A variety of approaches can be used to minimize the effects of this spherical aberration. For example, a compensator can be used, such as described in U.S. Patent No. RE48,561, which is hereby incorporated by reference in its entirety. However, for some systems, the aberration may be so significant that the expense and/or complexity of a compensator-based system may be a barrier. Therefore, in some embodiments, in addition to (or as an alternative to) incorporating a compensator into the imaging system, the aberration can be accounted for by coordinating the choice of immersion fluid and the movement of the objective lens 92 between imaging at different surfaces (e.g., the top and bottom surfaces) so that the value of Δ in Equation 3 decreases to zero, taking into account the values of _nb , _tb , and _θb associated with the system. To illustrate, consider the case where the immersion fluid is chosen to have the same refractive index as the reagent solution in the interior volume 46 of the support structure 16 (e.g., n _b = 1.36). This can be accomplished, for example, in systems that use the same fluid as the buffer fluid and the immersion fluid and keep the temperature of those fluids constant; systems that use water with salt or other organic compounds dissolved to obtain the appropriate refractive index; or other types of systems (e.g., systems that use matching liquids). In this case, n _b will be equal to n _i , and θ _b will be equal to θ _i , allowing Equation 5 to be simplified to Equation 4 below.

Using this equation, a Δ value of 0 (i.e., identical image quality for the bottom and top surfaces) can be achieved by setting _ti ' equal to _ti - _tb . Thus, the imaging system can achieve diffraction-limited image quality for both the top and bottom surfaces of a flow cell by performing a first imaging pass of the top surface with the objective lens separated from the cover glass by a distance _ti , using an immersion fluid with the same refractive index as its reagent solution, and then performing a second imaging pass of the bottom surface with the objective lens separated from the cover glass by a distance equal to _ti minus _tb .

In addition to the compensator-based approach described in RE48,561, or matching _nb and _ni and setting _ti ' to reduce Δ to zero, other possible approaches are possible. For example, in some cases, rather than setting the difference between nb and ni to zero, systems based on the present disclosure use an immersion fluid with a value of _ni that is within a certain tolerance range _{of nb} , so that sufficient image quality can still be achieved under the specific circumstances of the system application under consideration. To illustrate how this type of approach can be implemented, consider a system with a numerical aperture of 1.2, which can achieve _diffraction -limited performance as long as the combined primary and higher-order spherical aberrations are less than or equal to 0.07 waves. In this case, the high- and low-order spherical aberrations generated by the mismatch between the refractive indices of the buffer and immersion fluids can be calculated by applying Zernike analysis using Equations 5 and 6 below.

In Equations 5 and 6, ρ is the value of sin(ψ) in the unit circle, ψ is the angle equal to θb - θi, and c _qm corresponds to the Zörne radial variable. ( ρ ) is the Zörneke expansion coefficient, and δm0 is a normalized variable (equal to ₁ if m is 0 and 0 otherwise). Applying this analysis to an exemplary 1.2NA system provides the primary and high-order spherical aberration information listed in Table 1 below for various illustrative theoretical immersion fluids having a specific refractive index difference (i.e., Δn) with the buffer fluid used in the system.

In this example, diffraction-limited behavior is achieved as long as the refractive index difference between the buffer and immersion fluids does not exceed 0.0003, meaning that immersion fluids G, H, I, or J can be used, despite the fact that only immersion fluid J has the same refractive index as the buffer fluid used in the system.

As another type of variation, although the examples above discuss systems in which the objective is configured to compensate for expected aberrations during imaging of the top surface and then the manipulable parameters are chosen to match the bottom surface image, the objective can also be configured to compensate for expected aberrations during imaging of the bottom surface and the manipulable parameters are chosen to match the top surface image to these aberrations. The above approach can also be applied to more than two surfaces, for example by selecting the immersion fluid and manipulating the position of the objective between imaging operations so that the difference between the axial and marginal rays is the same for each surface. Other variations are possible (e.g., setting the system's manipulable parameters to sufficiently reduce aberrations to allow for the use of less complex compensators, rather than setting those parameters to completely eliminate the need for compensators) and will be apparent to those skilled in the art with reference to this disclosure. Therefore, the above description of the method for addressing aberration differences between surfaces and its exemplary variations should be understood as illustrative only and not limiting.

In certain embodiments, systems based on implementations of the present disclosure can utilize sequencing-by-synthesis (SBS). In SBS, one or more fluorescently labeled modified nucleotides are used to determine the nucleotide sequence of a nucleic acid present on the surface of a support structure, such as a flow cell. Exemplary SBS systems and methods that may be used with the apparatus and methods described herein are described in U.S. Patent No. 7,057,026; U.S. Patent Application Publication Nos. 2005/0100900, 2006/0188901, 2006/0240439, 2006/0281109, and 2007/0166705; and PCT Publication Nos. WO 05/065814, WO 06/064199, and WO 07/010251; each of which is incorporated herein by reference.

In a specific application of the apparatus and methods herein, a flow cell containing arrayed nucleic acids is processed through several repetitive cycles of an overall sequencing process. The nucleic acids are prepared so as to include oligonucleotide primers adjacent to an unknown target sequence. To initiate the first SBS sequencing cycle, one or more differently labeled nucleotides and a DNA polymerase are flowed into the flow cell. Single nucleotides can be added at a time, or the nucleotides used in the sequencing process can be specifically designed to have reversible termination properties so that each cycle of the sequencing reaction can occur simultaneously in the presence of all four labeled nucleotides (A, C, T, G). After the nucleotides are added, features on the surface can be imaged to determine the identity of the incorporated nucleotide (based on the label on the nucleotide). Next, reagents can be added to the flow cell to remove the blocked 3' end (if applicable) and the label from each incorporated base. These cycles are then repeated, and the sequence of each population is read over multiple chemical cycles. In further embodiments, fluorinated nucleotides or other detectable moieties can be temporarily associated with the unknown target sequence to allow for an imaging step that is separate from the process of advancing to the next nucleotide.

Other sequencing methods that use cyclic reactions, where each cycle includes a step of delivering one or more reagents to nucleic acids on a surface and imaging the nucleic acids bound to the surface, such as pyrosequencing and sequencing by ligation, can also be used. Suitable pyrosequencing reactions are described, for example, in U.S. Patent No. 7,244,559 and U.S. Patent Application Publication No. 2005/0191698, each of which is incorporated herein by reference. Zygosity sequencing reactions are described, for example, in Shendure et al., Science 309: 1728-1732 (2005); and U.S. Patent Nos. 5,599,675 and 5,750,341, each of which is incorporated herein by reference.

The methods and apparatus described herein are also suitable for detecting surface-appearing features used in genotyping assays, phenotyping assays, and other assays known in the art (e.g., as described in U.S. Patent Application Publication Nos. 2003/0108900, 2003/0215821, and 2005/0181394, each of which is incorporated herein by reference).

While various embodiments of the present disclosure have been described above, it should be understood that these are presented by way of example only and are not limiting. Similarly, the various figures may depict example architectures or other configurations of the present disclosure, which are provided to facilitate understanding of the features and functionality that may be included in the present disclosure. The present disclosure is not limited to the example architectures or configurations depicted, but various alternative architectures and configurations may be used to implement desired features. In practice, it will be apparent to those skilled in the art how to implement alternative functional, logical or physical partitioning and configurations to implement the desired features of the present disclosure. Furthermore, a variety of different component module names, in addition to those depicted herein, may be applied to the various partitions. Additionally, with respect to flow charts, operational descriptions, and method claims, the order in which the actions are presented herein should not imply that the various embodiments must be implemented in the same order to perform the described functions, unless the context indicates otherwise.

801: Top surface 802: Bottom surface

Claims (20)

An apparatus for a multi-surface biological sample imaging system comprises: a lens having a field of view and immersed in a first fluid having a first refractive index; a flow channel comprising a first surface and a second surface separated by a second fluid having a second refractive index; and a controller configured to: move the lens from a first position having a first distance from the flow channel to a second position having a second distance from the flow channel; when the lens is separated from the flow channel by the first distance, use the lens to capture light emitted by nucleic acids disposed on the first surface of the flow channel; when the lens is separated from the flow channel by the second distance, use the lens to capture light emitted by nucleic acids disposed on the second surface of the flow channel; and Determining a nucleic acid sequence in a biological sample based on the emitted light; wherein, when the lens is separated from the flow channel by the first distance, an optical path difference between marginal light and axial light in the field of view of the lens is substantially equal to an optical path difference between marginal light and axial light in the field of view of the lens when the lens is separated from the flow channel by the second distance. The apparatus of claim 1, wherein the first refractive index and the second refractive index are substantially equal. The apparatus of claim 2, wherein: the lens is comprised of an optical device adapted to capture light emitted by nucleic acid sequences disposed on the first surface of the flow cell and to allow the biological sample to be imaged on a detector with diffraction-limited imaging quality; and the first refractive index and the second refractive index are substantially equal, meaning that any spherical aberration caused by any difference between the first refractive index and the second refractive index is sufficiently low as not to prevent diffraction-limited imaging of the biological sample based on: light emitted by nucleic acid sequences disposed on the first surface of the flow cell; and light emitted by nucleic acid sequences disposed on the second surface of the flow cell. The machine of claim 2, wherein the first fluid and the second fluid are the same fluid. The machine of claim 2, wherein the first fluid and the second fluid are different fluids. The machine of claim 1 , wherein the first surface of the flow channel is a top surface of the flow channel, and the second surface of the flow channel is a bottom surface of the flow channel. The apparatus of claim 1, wherein: the lens is comprised of an optical device adapted to capture light emitted by nucleic acid sequences disposed on the first surface of the flow cell and to allow imaging of the biological sample on a detector with diffraction-limited imaging quality; and the optical path difference between marginal and axial rays in the field of view of the lens when the lens is separated from the flow cell by the first distance is substantially equal to the optical path difference between marginal and axial rays in the field of view of the lens when the lens is separated from the flow cell by the second distance, i.e., spherical aberration is sufficiently low so as not to prevent diffraction-limited imaging of the biological sample based on: Light emitted by nucleic acid sequences disposed on the first surface of the flow cell; and Light emitted by nucleic acid sequences disposed on the second surface of the flow cell. A method for imaging a multi-surface biological sample comprises: capturing light emitted by nucleic acids disposed on a first surface of a flow channel using a lens having a field of view, the lens being: at a first distance from the flow channel; and immersed in a first fluid having a first refractive index; moving the lens to a position at a second distance from the flow channel; capturing light emitted by nucleic acids disposed on a second surface of the flow channel using the lens, the lens being immersed in the first fluid having the first refractive index, and the lens being at the second distance from the flow channel, wherein the first surface of the flow channel is separated from the second surface of the flow channel by a second fluid having a second refractive index; and Determining a nucleic acid sequence in a biological sample based on the light emitted by nucleic acids disposed on the first surface of the flow channel and the light emitted by nucleic acids disposed on the second surface of the flow channel; wherein, when the lens is at the first distance from the flow channel, an optical path difference between marginal light and axial light in the field of view of the lens is substantially equal to an optical path difference between marginal light and axial light in the field of view of the lens when the lens is at the second distance from the flow channel. The method of claim 8, wherein the first refractive index and the second refractive index are substantially equal. The method of claim 9, wherein: the lens is comprised of an optical device adapted to capture light emitted by nucleic acid sequences disposed on the first surface of the flow cell and to allow the biological sample to be imaged on a detector with diffraction-limited imaging quality; and the first refractive index and the second refractive index are substantially equal, meaning that any spherical aberration caused by any difference between the first refractive index and the second refractive index is sufficiently low as not to prevent diffraction-limited imaging of the biological sample based on: light emitted by nucleic acid sequences disposed on the first surface of the flow cell; and light emitted by nucleic acid sequences disposed on the second surface of the flow cell. The method of claim 9, wherein the first fluid and the second fluid are the same fluid. The method of claim 9, wherein the first fluid and the second fluid are different fluids. The method of claim 8, wherein the first surface of the flow channel is a top surface of the flow channel, and the second surface of the flow channel is a bottom surface of the flow channel. The method of claim 8, wherein: the lens is comprised of an optical device adapted to capture light emitted by nucleic acid sequences disposed on the first surface of the flow cell and to allow the biological sample to be imaged on a detector with diffraction-limited imaging quality; and the optical path difference between marginal and axial rays in the field of view of the lens when the lens is at the first distance from the flow cell is substantially equal to the optical path difference between marginal and axial rays in the field of view of the lens when the lens is at the second distance from the flow cell, i.e., spherical aberration is sufficiently low so as not to prevent diffraction-limited imaging of the biological sample based on: light emitted by nucleic acid sequences disposed on the first surface of the flow cell; and Light emitted by the nucleic acid sequence disposed on the second surface of the flow cell. A non-transitory computer-readable medium stores instructions that, when executed by a processor, cause a biological sample imaging system to perform actions, the actions comprising: capturing light emitted by nucleic acid disposed on a first surface of a flow channel using a lens, the lens being at a first distance from the flow channel, the lens having a field of view, and immersed in a first fluid having a first refractive index; moving the lens to a position at a second distance from the flow channel; capturing light emitted by nucleic acids disposed on a second surface of the flow cell using the lens, the lens being immersed in the first fluid having the first refractive index and the lens being at a second distance from the flow cell, wherein the first surface of the flow cell and the second surface of the flow cell are separated by a second fluid having a second refractive index; and determining a nucleic acid sequence in a biological sample based on the light emitted by the nucleic acids disposed on the first surface of the flow cell and the light emitted by the nucleic acids disposed on the second surface of the flow cell; When the lens is at the first distance from the flow channel, an optical path difference between a marginal ray and an axial ray in the field of view of the lens is substantially equal to an optical path difference between a marginal ray and an axial ray in the field of view of the lens when the lens is at the second distance from the flow channel. The non-transitory computer-readable medium of claim 15, wherein the first refractive index and the second refractive index are substantially equal. The non-transitory computer-readable medium of claim 16, wherein: the lens is comprised of an optical device adapted to capture light emitted by nucleic acid sequences disposed on the first surface of the flow cell and to allow the biological sample to be imaged on a detector with diffraction-limited imaging quality; and the first refractive index and the second refractive index are substantially equal, meaning that any spherical aberration caused by any difference between the first refractive index and the second refractive index is sufficiently low as not to prevent diffraction-limited imaging of the biological sample based on: light emitted by nucleic acid sequences disposed on the first surface of the flow cell; and light emitted by nucleic acid sequences disposed on the second surface of the flow cell. The non-transitory computer-readable medium of claim 15, wherein the first surface of the flow channel is a top surface of the flow channel, and the second surface of the flow channel is a bottom surface of the flow channel. The non-transitory computer-readable medium of claim 15, wherein: the lens is comprised of an optical device adapted to capture light emitted by nucleic acid sequences disposed on the first surface of the flow cell and to allow the biological sample to be imaged on a detector with diffraction-limited imaging quality; and the optical path difference between marginal and axial rays in the field of view of the lens when the lens is at the first distance from the flow cell is substantially equal to the optical path difference between marginal and axial rays in the field of view of the lens when the lens is at the second distance from the flow cell, i.e., spherical aberration is sufficiently low so as not to prevent diffraction-limited imaging of the biological sample based on: Light emitted by nucleic acid sequences disposed on the first surface of the flow cell; and Light emitted by nucleic acid sequences disposed on the second surface of the flow cell. The non-transitory computer-readable medium of claim 15, wherein the first fluid and the second fluid are the same fluid.